The present invention relates to a novel method for producing an N-acetyllactosamine oligosaccharide and more particularly to a method for producing an N-acetyllactosamine oligosaccharide by enzymatic reaction and chemical reaction from keratan sulfate whose sulfate group content has been adjusted. Also, the present invention relates to a novel method for producing a sulfated N-acetyllactosamine oligosaccharide. Further, the present invention relates to a novel N-acetyllactosamine oligosaccharide.
Oligosaccharides having an N-acetyllactosamine (LacNAc) structure as their skeleton and derivatives thereof play an important physiological role in vivo as a sugar chain having ability to bind to selectin family compounds. It has been revealed that expression of these sugar chains on cell surfaces participates in specific adhesion between heterogeneous cells, and function activators or inhibitors utilizing these sugar chains are expected to provide useful drugs. That is, sialyl Lewis X (SLex), which is a representative compound, and analogues thereof are considered to be useful for alleviating various diseases accompanied with inflammation by blocking selectin molecules to inhibit the adhesion of lymphocytes to endothelial cells. Further, these sugar chains are considered to participate in adhesion of cancer cells to endothelial cells when the cancer cells undergo blood circulatory metastasis or adhesion of microorganisms to target cells when the microorganisms infect to the target cells, and therefore attached importance as a raw material for developing drugs for suppressing these diseases.
The oligosaccharides having an N-acetyllactosamine structure as their skeleton and derivatives thereof are expected to play an important role in the development of novel drugs and in the research of physiological activity in vivo. If there is obtained an N-acetyllactosamine oligosaccharide, which is their basic skeleton, in particular a long chain N-acetyllactosamine oligosaccharide similar to natural-occurring ligand sugar chains of selectin family, it is considered possible to synthesize the ligand sugar chains of selectin family, which occurs in a trace amount in a natural state, by adding properly a sialic acid residue, a fucosyl residue, a sulfate group, ceramide or the like to the N-acetyllactosamine oligosaccharide.
Further, stable supply of N-acetyllactosamine oligosaccharides, in particular long chain N-acetyllactosamine oligosaccharides, is desired. However, oligosaccharides that have N-acetyllactosamine structure as their skeleton exist only in a trace amount in a natural state and it is very difficult to synthesize long chain N-acetyllactosamine oligosaccharides so that a method for producing N-acetyllactosamine oligosaccharides on an industrial scale and at low costs is desired.
Also, sulfated N-acetyllactosamine oligosaccharides, in particular long chain sulfated N-acetyllactosamine oligosaccharides, are useful as intermediates for the synthesis of N-acetyllactosamine oligosaccharides, in particular long chain N-acetyllactosamine oligosaccharides and, hence, it is expected that provision of a method for producing such oligosaccharides will lead to stable supply of N-acetyllactosamine oligosaccharides.
The present invention has been made from the above-described viewpoints, and its object is to provide a novel N-acetyllactosamine oligosaccharide which serves as a raw material for the synthesis of the ligand sugar chains of selectin family, a novel method for producing an N-acetyllactosamine oligosaccharide, and a novel method for producing a sulfated N-acetyllactosamine oligosaccharide.
As a result of intensive investigation by the present inventors with view to achieving the above-described object, it has now been found that an N-acetyllactosamine oligosaccharide (in particular a long chain N-acetyllactosamine oligosaccharide) can be obtained by adjusting the sulfate group content of keratan sulfate having an N-acetyllactosamine structure as a basic skeleton, enzymatically degrading the keratan sulfate with the thus adjusted sulfate group content to sulfated N-acetyllactosamine oligosaccharides, and then completely desulfating the sulfated N-acetyllactosamine oligosaccharides. The present invention has been completed based on this finding.
It has also been found that a sulfated N-acetyllactosamine oligosaccharide (in particular a long chain sulfated N-acetyllactosamine oligosaccharide) can be obtained by adjusting the sulfate group content of keratan sulfate and enzymatically digesting the keratan sulfate with the adjusted sulfate group content and the present invention has been completed based on this finding.
Further, it has been successful in obtaining a novel N-acetyllactosamine oligosaccharide, which has led to the present invention.
Thus, the present invention provides a method for producing an N-acetyllactosamine oligosaccharide, comprising the steps of: adjusting a sulfate group content of keratan sulfate, allowing an enzyme having an activity to cleave a glycosidic linkage of keratan sulfate (herein sometimes referred to simply as xe2x80x9ckeratan sulfate-degrading enzymexe2x80x9d) to act on the keratan sulfate with the adjusted sulfate group content to obtain a sulfated N-acetyllactosamine oligosaccharide, and then completely desulfating the sulfated N-acetyllactosamine oligosaccharide (herein sometimes referred to simply as xe2x80x9cmethod 1 of the present inventionxe2x80x9d), and a novel N-acetyllactosamine oligosaccharide (herein sometimes referred to simply as xe2x80x9coligosaccharide of the present inventionxe2x80x9d).
Also, the present invention provides a method for producing a sulfated N-acetyllactosamine oligosaccharide, comprising at least the steps of: adjusting a sulfate group content of keratan sulfate, and allowing one or more enzymes belonging to any one of the enzyme groups of three types of keratan sulfate-degrading enzymes each having a different substrate specificity depending on presence or absence of a sulfate group to act on the keratan sulfate with the adjusted sulfate group content (herein sometimes referred to simply as xe2x80x9cmethod 2 of the present inventionxe2x80x9d). Note that sometimes the methods 1 and 2 of the present invention are collectively referred to simply as xe2x80x9cmethod of the present inventionxe2x80x9d.
The term xe2x80x9cN-acetyllactosaminexe2x80x9d as used herein refers to a structure (Gal-GlcNAc) containing a galactose residue (Gal) and an N-acetylglucosamine residue (GlcNAc) linked through a glycosidic linkage. Also, the term xe2x80x9cN-acetyllactosaminexe2x80x9d as used herein includes a structure (GlcNAc-Gal) containing an N-acetylglucosamine residue (GlcNAc) and a galactose residue (Gal) are linked through a glycosidic linkage.
The term xe2x80x9cN-acetyllactosamine oligosaccharidexe2x80x9d as used herein refers to an oligosaccharide having at least one N-acetyllactosamine structure, and includes, for example, N-acetyllactosamine itself and oligosaccharides containing N-acetyllactosamine units linked one after another repeatedly through a glycosidic linkage. Also, a sialic acid residue may be added to its non-reducing ends. The non-reducing end and reducing end may be either a galactose residue or an N-acetylglucosamine residue. These are both embraced by the term xe2x80x9cN-acetyllactosamine oligosaccharidexe2x80x9d used herein. That is, the oligosaccharides represented by the following general formulae (1) to (6) are encompassed by the xe2x80x9cN-acetyllactosamine oligosaccharidexe2x80x9d used herein:
(GlcNAc-Gal)nxe2x80x83xe2x80x83(1)
SA-Gal-(GlcNAc-Gal)mxe2x80x83xe2x80x83(2)
(Gal-GlcNAc)nxe2x80x83xe2x80x83(3)
SA-(Gal-GlcNAc)mxe2x80x83xe2x80x83(4)
Gal-(GlcNAc-Gal)nxe2x80x83xe2x80x83(5)
GlcNAc-(Gal-GlcNAc)nxe2x80x83xe2x80x83(6)
wherein, Gal represents a galactose residue, GlcNAc represents an N-acetylglucosamine residue, SA represents a sialic acid residue, xe2x80x94 represents a glycosidic linkage, n is an integer of 1 to 6, and m is an integer of 1 to 10.
The term xe2x80x9csulfated N-acetyllactosamine oligosaccharidexe2x80x9d as used herein refers to an N-acetyllactosamine oligosaccharide having a sulfate group.
The term xe2x80x9csulfated oligosaccharidexe2x80x9d as used herein refers to an oligosaccharide having a sulfate group.
The term xe2x80x9cadjusting a sulfate group contentxe2x80x9d as used herein refers to increasing a sulfate group content (i.e., sulfating), decreasing a sulfate group content (i.e., partially desulfating), and unchanging a sulfate group content if a desired sulfate group content has already been reached without further increasing or decreasing it.
The term xe2x80x9cpartially desulfatingxe2x80x9d as used herein refers to removing partially sulfate groups and differs from xe2x80x9ccompletely desulfatingxe2x80x9d in that the former allows a part of sulfate groups to remain.
The term xe2x80x9ccompletely desulfatingxe2x80x9d as used herein refers to removing substantially all the sulfate groups, thus differing from partially removing sulfate groups.